We have identified and characterized the cis-acting transcriptional control elements that interact with cellular and viral proteins to regulate equine infectious anemia virus (EIAV) gene expression. EIAV is a lentivirus distantly related to the human and simian immunodeficiency viruses (HIV and SIV). EIAV promoter sequences were inserted into a plasmid upstream of the chloramphenicol acetyltransferase (CAT) gene; promoter activity was quantified by analysis of CAT mRNA or CAT enzyme activity. We first defined the cis-acting element that mediates transcriptional activation by the viral Tat protein. The EIAV Tat- response element (TAR) was shown to be a 25 nucleotide stem-loop structure located at the 5' end of EIAV RNA. Mutations that disrupted the hairpin structure abolished trans-activation; compensatory mutations that restored the hairpin, but altered the primary sequence, restored activity. Although EIAV TAR is function-ally analogous to HIV-1 TAR, it is structurally distinct. Interactions of cellular DNA-binding proteins with the EIAV promoter were examined by DNase footprinting and gel mobility shift assays. In addition, specific elements were functionally identified by mutagenesis and transient transfections. Several discrete elements were defined including elements that bind to the transcription factors PEA-2, AP-1, OCT, ets, and PU.1. The protein PU.1 is identical to the oncogene Spi-1 whose expression is restricted to monocytes, macrophages, and B-lymphocytes.